Recently, an increased effort has been directed toward the development of chips for molecular detection. In general, a molecular detection chip includes a substrate on which an array of binding sites is arranged. Each binding site (or hybridization site) has a respective molecular receptor (or probe) which binds or hybridizes with a molecule having a predetermined structure. A sample solution is applied to the molecular detection chip, and molecules in the sample bind or hybridize at one or more of the binding sites. The particular binding sites at which hybridization occurs are detected, and one or more molecular structures within the sample are subsequently deduced.
Of great interest are molecular detection chips for gene sequencing. These chips, often referred to as DNA chips, utilize an array of selective binding sites each having respective single-stranded DNA probes. A sample of single-stranded DNA fragments, referred to as target DNA, is applied to the DNA chip. The DNA fragments attach to one or more of the DNA probes by a hybridization process. By detecting which DNA probes have a DNA fragment hybridized thereto, a sequence of nucleotide bases within the DNA fragment can be determined.
Various approaches have been utilized to detect a hybridization event at a binding site. Many different approaches such as optical and electrical detection have been developed, but only electrical detection will be addressed herein.
The specific approach of interest is electrochemical based detection. In this approach a standard three-electrode potentiostat including an auxiliary electrode, a reference electrode and a working electrode is provided. DNA probes are covalently bonded to the surface of the working electrode by linker groups. Exposure of the single stranded DNA probes to complementary single-stranded DNA in solution will result in a hybridization reaction specifically dictated by the DNA base sequence. By applying voltemetric measurement techniques, DNA hybridization reactions are measured by observing the change in peak current caused by an oxidation/reduction reaction. This reaction is completed by introducing an intercalator that specifically binds to the minor groove of double stranded DNA through electrostatic interactions. As a result, the current peak due to oxidation/reduction of the intercalator bound to the hybridized DNA will indicate the hybridization event.
The problem with detecting a current peak associated with the oxidation/reduction reaction of the intercalator at the hybridization event is background current. The sensitivity of these types of devices is limited because intercalators may have an oxidation/reduction reaction with the working electrode surface, resulting in background current. Furthermore, while the intercalators have a tendency to bind to the minor groove of a double stranded DNA molecule through electrostatic interactions, they will also bind to a single stranded DNA, although not as readily. This further reduces sensitivity.
It would be highly advantageous, therefore, to remedy the foregoing and other deficiencies inherent in the prior art.
Accordingly, it is an object of the present invention to provide a new and improved method and apparatus for identifying molecules.
Another object of the present invention is to provide a new and improved method and apparatus for identifying molecules employing electrochemical detection.
And another object of the present invention is to provide a new and improved method and apparatus for electrochemical detection of molecules, having reduced background current.
Yet another object of the present invention is to provide a new and improved method and apparatus for electrochemical detection of molecules, having increase sensitivity.
Still another object of the present invention is to provide a new and improved method and apparatus for electrical detection of molecules, employing a probe which limits the attraction of intercalators thereto.